Correction for Fletcher et al., "Vaccine Potential of the Neisseria meningitidis 2086 Lipoprotein".

[This corrects the article DOI: 10.1128/IAI.72.4.2088-2100.2004.].

NMR dynamics and antibody recognition of the meningococcal lipidated outer membrane protein LP2086 in micellar solution.

Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.

The discovery and development of a novel vaccine to protect against Neisseria meningitidis Serogroup B Disease.

Vaccines have had a major impact on the reduction of many diseases globally. Vaccines targeted against invasive meningococcal disease (IMD) due to serogroups A, C, W, and Y are used to prevent these diseases. Until recently no vaccine had been identified that could confer broad protection against Neisseria meningitidis serogroup B (MnB). MnB causes IMD in the very young, adolescents and young adults and thus represents a significant unmet medical need. In this brief review, we describe the discovery and development of a vaccine that has the potential for broad protection against this devastating disease.

Role of factor H binding protein in Neisseria meningitidis virulence and its potential as a vaccine candidate to broadly protect against meningococcal disease.

Neisseria meningitidis is a Gram-negative microorganism that exists exclusively in humans and can cause devastating invasive disease. Although capsular polysaccharide-based vaccines against serogroups A, C, Y, and W135 are widely available, the pathway to a broadly protective vaccine against serogroup B has been more complex. The last 11 years has seen the discovery and development of the N. meningitidis serogroup B (MnB) outer membrane protein factor H binding protein (fHBP) as a vaccine component. Since the initial discovery of fHBP, a tremendous amount of work has accumulated on the diversity, structure, and regulation of this important protein. fHBP has proved to be a virulence factor for N. meningitidis and a target for functional bactericidal antibodies. fHBP is critical for survival of meningococci in the human host, as it is responsible for the primary interaction with human factor H (fH). Binding of hfH by the meningococcus serves to downregulate the host alternative complement pathway and helps the organism evade host innate immunity. Preclinical studies have shown that an fHBP-based vaccine can elicit serum bactericidal antibodies capable of killing MnB, and the vaccine has shown very encouraging results in human clinical trials. This report reviews our current knowledge of fHBP. In particular, we discuss the recent advances in our understanding of fHBP, its importance to N. meningitidis, and its potential role as a vaccine for preventing MnB disease.

Broad vaccine coverage predicted for a bivalent recombinant factor H binding protein based vaccine to prevent serogroup B meningococcal disease.

Factor H binding proteins (fHBP), are bacterial surface proteins currently undergoing human clinical trials as candidate serogroup B Neisseria meningitidis (MnB) vaccines. fHBP protein sequences segregate into two distinct subfamilies, designated A and B. Here, we report the specificity and vaccine potential of mono- or bivalent fHBP-containing vaccines. A bivalent fHBP vaccine composed of a member of each subfamily elicited substantially broader bactericidal activity against MnB strains expressing heterologous fHBP than did either of the monovalent vaccines. Bivalent rabbit immune sera tested in serum bactericidal antibody assays (SBAs) against a diverse panel of MnB clinical isolates killed 87 of the 100 isolates. Bivalent human immune sera killed 36 of 45 MnB isolates tested in SBAs. Factors such as fHBP protein variant, PorA subtype, or MLST were not predictive of whether the MnB strain could be killed by rabbit or human immune sera. Instead, the best predictor for killing in the SBA was the level of in vitro surface expression of fHBP. The bivalent fHBP vaccine candidate induced immune sera that killed MnB isolates representing the major MLST complexes, prevalent PorA subtypes, and fHBP variants that span the breadth of the fHBP phylogenetic tree. Importantly, epidemiologically prevalent fHBP variants from both subfamilies were killed.

Structural Basis for the Immunogenic Properties of the Meningococcal Vaccine Candidate LP2086.

LP2086 is a family of outer membrane lipoproteins from Neisseria meningitidis, which elicits bactericidal antibodies and are currently undergoing human clinical trials in a bivalent formulation where each antigen represents one of the two known LP2086 subfamilies. Here we report the NMR structure of the recombinant LP2086 variant B01, a representative of the LP2086 subfamily B. The structure reveals a novel fold composed of two domains: a "taco-shaped" N-terminal beta-sheet and a C-terminal beta-barrel connected by a linker. The structure in micellar solution is consistent with a model of LP2086 anchored to the outer membrane bilayer through its lipidated N terminus. A long flexible chain connects the folded part of the protein to the lipid anchor and acts as spacer, making both domains accessible to the host immune system. Antibodies broadly reactive against members from both subfamilies have been mapped to the N terminus. A surface of subfamily-defining residues was identified on one face of the protein, offering an explanation for the induction of subfamily-specific bactericidal antibodies.

Detection of LP2086 on the cell surface of Neisseria meningitidis and its accessibility in the presence of serogroup B capsular polysaccharide.

The outer membrane protein LP2086, a human factor H binding protein, is undergoing clinical trials as a vaccine against invasive serogroup B meningococcal (MnB) disease. As LP2086 is a surface protein, expression of capsular polysaccharide could potentially limit accessibility of anti-LP2086 antibodies to LP2086 expressed on the surface of bacteria. To determine whether variability in expression levels of the serogroup B capsule (Cap B) might interfere with accessibility of anti-LP2086 antibody binding to LP2086, we evaluated the ability of anti-Cap B and anti-LP2086 antibodies to bind to the surface of 1263 invasive clinical MnB strains by flow cytometry. One of the anti-LP2086 monoclonal antibodies used recognizes virtually all LP2086 sequence variants. Our results show no correlation between the amount of Cap B expressed and the binding of anti-LP2086 antibodies. Furthermore, the susceptibility of MnB bacteria to lysis by anti-LP2086 immune sera was independent of the level of Cap B expressed. The data presented in this paper demonstrates that Cap B does not interfere with the binding of antibodies to LP2086 expressed on the outer membrane of MnB clinical isolates.

Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria meningitidis.

BACKGROUND: Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. METHODS: Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. RESULTS: Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. CONCLUSIONS: The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.

Certain site-directed, nonenzymatically active mutants of the Haemophilus influenzae P4 lipoprotein are able to elicit bactericidal antibodies.

The Haemophilus influenzae P4 lipoprotein (hel) is a potential component of a nontypeable H. influenzae otitis media vaccine. Since P4 is known to be an enzyme, nonenzymatically active forms of recombinant P4 are required. After site-directed mutagenesis of the hel gene, three of the mutated proteins were shown to be vaccine candidates.

Reduction of nasal colonization of nontypeable Haemophilus influenzae following intranasal immunization with rLP4/rLP6/UspA2 proteins combined with aqueous formulation of RC529.

Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis are common causative agents of human mucosal infections. To formulate a mucosal vaccine against these pathogens, recombinant lipidated P4 (rLP4) and P6 (rLP6) proteins of NTHi and ubiquitous cell surface protein A (UspA) of M. catarrhalis were used for active immunization experiments in a mouse nasal challenge model. BALB/c mice were immunized intranasally with these proteins formulated with a chemically synthesized adjuvant, RC529 in an aqueous formulation (RC529-AF). Three weeks after the last immunization, these animals were challenged intranasally with NTHi strain SR7332.P1 and nasal colonization measured 3 days later. To determine local and systemic immune responses, bronchoalveolar washes (BAW) and sera were collected prior to NTHi challenge. The serum and mucosal samples were analyzed by ELISA for rLP4, rLP6 and UspA2 protein-specific IgG, IgG subclass and IgA antibody titers and bactericidal titers were determined against the TTA24 and 430-345 strains of M. catarrhalis. Results of these experiments show that these proteins combined with RC529-AF administered intranasally to mice elicited (1) significantly increased rLP4/rLP6/UspA2 protein-specific circulating IgG and IgA antibody responses; (2) local rLP4/rLP6/UspA2-specific IgA responses in the respiratory tract; and (3) more than a two log reduction of nasal colonization of NTHi strain SR7332 from the nasal tissues of mice. The serum IgG subclass distribution was predominantly IgG2a, representing a Th1 response. The antiserum also exhibited bactericidal activities to several strains of M. catarrhalis. These data indicate that intranasal immunization with rLP4/rLP6/UspA2 proteins combined with RC529-AF may be able to provide a way for inducing local mucosal immunity and for prevention of otitis media in children.

Vaccine potential of the Neisseria meningitidis 2086 lipoprotein.

A novel antigen that induces cross-reactive bactericidal antibodies against a number of Neisseria meningitidis strains is described. This antigen, a approximately 28-kDa lipoprotein called LP2086, was first observed within a complex mixture of soluble outer membrane proteins (sOMPs) following a series of fractionation, protein purification, and proteomics steps. Approximately 95 different neisserial isolates tested positive by Western blotting and PCR screening methods for the presence of the protein and the gene encoding LP2086. The strains tested included isolates of N. meningitidis serogroups A, B, C, W135, and Y, Neisseria gonorrhoeae, and Neisseria lactamica. To better understand the microheterogeneity of this protein, the 2086 genes from 63 neisserial isolates were sequenced. Two different subfamilies of LP2086 were identified based on deduced amino acid sequence homology. A high degree of amino acid sequence similarity exists within each 2086 subfamily. The highest degree of genetic diversity was seen between the two subfamilies which share approximately 60 to 75% homology at the nucleic acid level. Flow cytometry (fluorescence-activated cell sorting) analyses and electron microscopy indicated that the LP2086 is localized on the outer surface of N. meningitidis. Antiserum produced against a single protein variant was capable of eliciting bactericidal activity against strains expressing different serosubtype antigens. Combining one recombinant lipidated 2086 (rLP2086) variant from each subfamily with two rPorA variants elicited bactericidal activity against all strains tested. The rLP2086 family of antigens are candidates worthy of further vaccine development.